Cystic Fibrosis (CF) is the most common fatal genetic disease in humans (Boat et al. (1989), Cystic Fibrosis, In: THE METABOLIC BASIS OF INHERITED DISEASE, Scriver, Beaudet, Sly and Valle, eds., McGraw Hill, New York, pp. 2649-2860). Based on both genetic and molecular analysis, a gene associated with CF was isolated as part of 21 individual cDNA clones and its protein product predicted (Kerem et al. (1989), Science 245: 1073-1080; Riordan et al. (1989), Science 245: 1066-1073; Rommens et al. (1989), Science 245: 1059-1065).
The product of the CF-associated gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is a protein of approximately 1480 amino acids made up of two repeated elements, each having six transmembrane segments and a nucleotide binding domain. The two repeats are separated by a large, polar, so-called R-domain containing multiple potential phosphorylation sites. Based on its predicted domain structure, CFTR is a member or a class of related proteins which includes the multi-drug resistance (MDR) or P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan et al., supra; Hyde et al. (1990), Nature 346: 362-365). Proteins in this group, characteristically, are involved in pumping molecules into or out of cells.
CFTR has been postulated to regulate the outward flow of anions from epithelial cells in response to phosphorylation by cyclic AMP-dependent protein kinase or protein kinase C (Riordan et al., supra; Frizzell et al., supra.; Welsh and Liedtke (1986), Nature 322: 467; Li et al. (1988), Nature 331: 358-360; Hwang et al. (1989), Science 244: 1351-1353).
Sequence analysis of the CF associated gene has revealed a variety of mutations (Cutting et al. (1990a), Nature 346: 366-369; Cutting et al. (1990b), Am. J. Hum. Genet. 47: 213; Dean et al. (1990), Cell 61: 863-870; Kerem et al. (1989), Science 245: 1073-1080; and Kerem et al. (1990), Proc. Natl. Acad. Sci., USA 87: 8447-8451). Mutations in the gene encoding CFTR result in the synthesis of aberrant variants that are either unstable, mislocalized, or whose Cl.sup.- channel activity is dysfunctional as a consequence of defective regulation or conduction (Welsh and Smith (1993), Cell 73: 1251-1254). Over 200 different mutations have been described to date, but by far the most prevalent is a deletion of the three nucleotides that encode phenylalanine at position 508 (Phe.sup.508) located within the first nucleotide binding domain of CFTR (Tsui, L. C. (1992), Hum. Mutat. 1: 197-203). The Phe.sup.508 deletion (.DELTA.F508) is associated with approximately 70% of the cases of cystic fibrosis.
Studies on the biosynthesis (Cheng et al. (1990), Cell 63: 827-834; Gregory et al. (1990), Nature 347: 382-386) and localization (Denning et al. (1992), J. Cell Biol. 118: 551-559) of .DELTA.F508, as well as other CFTR mutants, indicate that many CFTR mutant proteins are not processed correctly and, as a result, are not delivered to the plasma membrane (Gregory et al., supra). These conclusions are consistent with earlier functional studies which failed to detect cAMP stimulated Cl.sup.- channels in cells expressing CFTR .DELTA.F508 (Rich et al., supra; Anderson et al. (1991), Science 251: 679-682).
It is believed that the deletion of residue 508 in .DELTA.F508-CFTR prevents the nascent protein from folding correctly, and consequently the variant is recognized by the quality control mechanism present within the endoplasmic reticulum (ER) to select out against misfolded or mutant proteins (Cheng et al. (1990), supra.; Gregory et al. supra.). The mutant .DELTA.F508-CFTR bears carbohydrate structures characteristic of glycosylation at the ER and is eventually degraded. The inability of this mutant protein to exit the ER, to pass through the Golgi where it normally would be fully glycosylated, and traffic to the plasma membrane most likely accounts for the defective Cl.sup.- transport found in CF epithelia harboring this mutation (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studies have shown, however, that .DELTA.F508-CFTR, when presented at the plasma membrane is functional as a cAMP-responsive Cl.sup.- channel (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al., supra.; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50).
Thus, there is a need in the art for methods and compositions which enable relocation of mislocalized CFTR mutants which retain at least some functional activity (i.e., .DELTA.F508) to the plasma membrane of epithelial cells where they can effectively mediate chloride ion transport and restore sufficient membrane conductance. The present invention satisfies this need and provides related advantages as well.